Development of fluorescently labeled cell surface markers has led to a rapid advancement in specific molecular diagnosis of diseases. These molecules can target specific cell receptors, important for diagnostics and choice of therapy. One example of such receptor is a well-known biomarker HER2 (human epidermal growth factor receptor type 2)that plays an important role in aggressive tumor behavior and is associated with poor clinical outcome. Applications of HER2-specific fluorescence probes are not limited to in vitro/ex vivo assays. These probes can potentially be used for in vivo imaging aimed at characterization/monitoring of HER2, because after injection specific fluorescent probes concentrate preferentially at the sites of interest (e.g., in the tumor), providing quantitative information on HER2 expression in vivo. Probe accumulation at the tumor cells can occur in parallel to the delivery of some widely used HER2 - specific drugs, such as, Trastuzumab, to the diseased site. Thus it opens a new era of treat and image paradigm. Assessment of HER2 expression in individual patients would facilitate selection of an optimal treatment strategy (for example, using monoclonal antibodies MAB), while the continuous monitoring of the status of those biomarkers during the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. Current techniques for estimating HER2 receptor expression use ex vivo assays that require tissue biopsies. They are not compatible with continuously monitoring of the response of the tumor receptors to the therapy. To the contrary, non-invasive in-vivo methods would be preferential, especially at early stages of the disease. We have developed a novel noninvasive method to characterize HER2 expression in-vivo, using optical imaging and HER2 - specific probes that could be used simultaneously with HER2 targeted therapies. HER2-specific near infrared fluorescent probe, based on small Affibody molecules, in combination with in-house time-resolved near-infrared fluorescence imaging system were used to characterize in-vivo tumors with varying levels of HER2 overexpression in the cancer cells. We tested this novel HER2 Affibody, conjugated to the fluorophore DyLight 750, (mouse model of the breast carcinoma) and showed that HER2 overexpression can be estimated from analysis of a series of the tumor fluorescence images, if the probe pharmacokinetics is taken into account by a ligand-receptor kinetic model. The new probe has high affinity to HER2 receptors (KD = 3.660.26). No acute toxicity observed from injection of the probes (up to 0.5 mg/kg) into mice. Our measurements have shown that after 1 min, the probe concentration in blood circulation decreases with time exponentially, with the average half-life washout time of 37 min from blood which is about 65 times faster than half-life of ABD-(ZHER2:342)2-AlexaFluor750 (40 hours), that was used in our previous studied. The short washout-time of this probe from blood circulation makes this probe a good candidate for potential clinical studies. The ligand-receptor kinetic model was applied to characterize the HER2 expression in three breast cancer xenograft models, expressing different levels of HER2. HER2 specific Affibody conjugated to DyLight750 were injected intravenously in mice bearing HER2 positive tumors, and the ROI was imaged at several predetermined time points. Based on pharmacokinetic studies average values of Normalized Rate of Accumulation (NRA) were calculated for each tumor type. They were compared with the Affibody-DyLight-488 retention measured by flow cytometry (FACS). The results indicate a good linear correlation between both parameters over the broad range of HER2 overexpression in cancer cells, substantiating our idea that in vivo fluorescence imaging can provide quantitative information on the HER2 overexpression Time-resolved fluorescence imaging system can provide additional information on the status of the tumor, if fluorescence lifetime is evaluated along with fluorescence intensity. The former parameter does not depend on the fluorophore concentration, but it can be sensitive to local biochemical environment of the probe, e.g., temperature, pH, or molecular interactions, provide potentially useful clinical information.In particular, we have demonstrated in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in-vivo. For this study HER2 specific Affibody probe, conjugated to Dylight 750, was injected intravenously in mice bearing HER2 positive and HER2 negative tumors and were imaged with our time resolved imaging system. We have compared fluorescence lifetimes of the optical probes in the bound (to tumor cells) and unbound state for different tumor types in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, lifetime measurements can be useful as a specific indicator of the receptor binding process, especially for early evaluation of the efficacy of the therapy. In collaboration with Washington University (Dr. Samuel Achilefu) and Imaging Probe Development Center at NIH, we have linked two pH sensitive dyes with malemide linkers. Previously the sensitivity of these dyes were tested in phantom and they showed different optical characteristics at different pH levels in the range of 5.5 to 7.0 (the pH range in normal tissue is 7.0-7.5 however, in many malignant lesions it can be as low as 5.6). These dyes will be conjugated with HER2 specific Affibody probes to further investigate their sensitivity in cells (in-vitro) and animals (in-vivo). We have also continued our work on using polaarization as a marker of structural changes.Polarization imaging allows one to separate contributions from surface specular reflection of the tissues and back scattered light from the deeper layers. As a result, hidden subsurface structures can be visualized to assess the transition of tissue from the healthy to diseased state. Correlation and filtering algorithms have been designed to improve image quality in the presence of noise and uncover regions of high statistical similarity, related, for example to the tissue collagen structure. To realize the potential of the method we have incorporated a user-friendly polarization imaging system into conventional colposcope. Based on our initial studies for 8 subjects, we realized that the surface of the cervix is not flat enough to get high quality cervix images without collecting data at multiple focal lengths. For this reason the polarization imaging system was upgraded to make possible automatic changes in focal length (stack of 10 co- and cross-polarized images at different focal planes is now being collected). We believe that an updated system is much more suitable for the clinical settings than the prototype and plan to continue our feasibility study to evaluate age related changes in the cervical structure at different time points of the menstrual cycle using polarization imaging. We have continued to evaluate diffuse multispectral imaging as a potentially useful supplement to existing response assessment in Kaposi Sarcoma, providing an early and non-invasive marker of treatment efficacy. Multi-spectral images of Kaposi Sarcoma skin lesions were taken over the course of treatment, and blood volume and oxygenation concentration maps were obtained through Principal Component Analysis (PCA) of the data. These images were compared with clinical and pathological responses determined by conventional means. We have found the oxygen ated hemoglobin has the potential to be a quantitative marker of tumor response.